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This research explores the minimizing of cracks in the fabrication of carbon-infiltrated carbon nanotube (CI-CNT) forests on cylindrical rods for the purposes of creating a CI-CNT coronary stent. It is a continuation of the work begun by Jones [1] and Skousen [2] whose work included the creation of a feasible CI-CNT coronary stent on a planar surface. The current research was performed in two parts: 1) growth on the whole circumference of the rod for ~20mm in length and 2) growth in lines and patterns. Experiments were done on either a 309 or 304 stainless steel rod ~3mm in diameter.The following parameters were used for growth on the whole circumference of the rod: a 80nm alumina diffusion barrier, 1.3 or 7nm of iron as a catalyst layer for CNT growth and a growth time of 1 or 15 min. Cracking is observed on most samples. Area fraction of cracking is shown to be minimized with 7nm of iron and and 1 min growth time. The number of cracks was minimized with the 7nm of iron and 15 min growth time. The height of the CI-CNT forest, which is strongly influenced by the thickness of the iron layer and growth time, is shown to be a possible positive contributor to the area fraction of cracking. Level of carbon infiltration and rate of cooling were also included in the parameters of the study as possible contributors to the amount of cracking however no correlation was found to either of these factors. The second study maintained 80nm of alumina and used 7nm of iron and 10 min of growth time. Line angles parallel, at 7°, at 45° and perpendicular to the axis of the rod and line widths from 109µm to 500µm were studied. Line widths from 500µm to 1090µm were also included in the study of patterned lines perpendicular to the axis of the rod. Cracking was eliminated in the parallel and 7° lines. Cracking in the 45° lines was intermittent and significantly less than the cracking seen on the whole circumference. For the lines perpendicular to the axis, the uncracked arc length was calculated to be an average of 414µm with a standard deviation of 67µm. The uncracked arc length showed no correlation to the line width. A final aspect of this second study observed the cracking in a modification of the planar stent pattern created by Jones. The pattern was modified in to allow for patterning on the rod. The critical dimension maximums of the parent pattern were maintained. The experimental results showed that a continual CI-CNT forest could be fabricated to the minimum expected height of 150µm, a maximum width of 542µm and at the expected angle of 7° with minimal to no cracking.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13207号 / 論医博第2161号 / 新制||医||1031(附属図書館) / (主査)教授 福原 俊一, 教授 湊谷 謙司, 教授 小池 薫 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The purpose of this research is to explore the potential of using carbon-infiltrated carbon nanotubes (CI-CNT) as a material for coronary artery stents. Stents are commonly fabricated from metal, which may not perform as well as many polymers and ceramics in biomedical applications. Pyrolytic carbon, a ceramic, is currently used in medical implant devices due to its preferrable biocompatibility properties. Micro-patterned pyrolytic carbon devices can be created by growing carbon nanotubes, and then filling the space between with amorphous carbon via chemical vapor deposition. We prepared multiple samples of two different planar stent-like flexible geometries and smaller cubic structures out of carbon infiltrated carbon nanotubes. These samples were tested in tension to failure. The cubic structures were used for separate compression tests. We also examined existing auxetic patterns for possible application in the stent designs and a second iteration of design and fabrication was performed using data and understanding obtained from the work in the first iteration. Slight changes were made to the mask design and fabrication processes based on the new geometries and testing considerations. The auxetic planar designs were tested in compression to demonstrate flexibility and collect material data. The testing results show that CI-CNTs can be designed and fabricated into flexible geometries capable of stent-like compression. The samples in this work were found to have moduli ranging from 5 to 27 GPa, with the majority being between 10 and 20 GPa. We also found fracture strength greater than 100 MPa, with it sometimes getting as high as 200 MPa. Lastly, fracture strain values were measured, with the maximum reaching 1.4% and the average between 0.75-1%. We also found that the CI-CNTs material lends itself to fracture at weak locations (if present) before the anticipated fracture strength has been reached and concluded that a tightly controlled process (including fabrication machines) environment is necessary to ensure consistent results and a CI-CNT material whose imperfections have been minimized.

The purpose of this research was to design, develop, and test coronary stent designs composed of carbon-infiltrated carbon nanotubes (CI-CNTs). Coronary stents currently have two major complications: restenosis and thrombosis. CI-CNT stents have potential to address both of these issues, and therefore may provide improved clinical outcomes. CI-CNT stent geometry is patterned using high-resolution photolithography that provide advantages in design possibilities.To develop a coronary stent, a standard design process was followed including: background, design specifications, concept generation, development, analysis, and testing. Background research was first completed and general design specifications for coronary stent performance were compiled. Multiple design concepts were generated, evaluated, and finally a design was selected. This stent design was further developed and optimized using analytical tools along with finite element analysis. This stent design used tapered struts in repeating segments to reduce stress and improve radial force. The design was modeled and analyzed as both a flat geometry as well as in a cylindrical configuration. Mechanics of materials equations and geometry specific finite element analysis were used to guide the final coronary stent design. The stent design was tested mechanically, and additional tests were performed to verify the blood compatibility of the CI-CNT material. The flat version of the stent design was manufactured and mechanically tested to verify performance. The performance of the cylindrical stent configuration was analyzed using an FE model of an atherosclerotic artery. This arterial FE model was created and validated by analyzing balloon angioplasty of a common stainless steel stent. The biocompatibility of CI-CNTs was explored and studied. Blood compatibility testing of CI-CNT samples was performed with results comparable in performance to stainless steel. A method of stent deployment was planned, and several other stent design concepts were analyzed. This research demonstrates that a functioning coronary stent can be manufactured from CI-CNTs. The optimized design has potential to address problems currently associated with stents. However, a major challenge for CI-CNT stent designs is meeting the design requirement of sufficient radial force. CI-CNT stents also need to have excellent blood compatibility to justify being used in stent applications.

<p>Accurately assessing the risk and growth of rupture among intracranial aneurysms (IA) remains a challenging task for clinicians. Hemodynamic factors are known to play a critical role in the development of IAs, but the specific mechanisms are not well understood. Many studies have sought to correlate specific flow metrics to risk of growth and rupture but have reported conflicting findings. Computational fluid dynamics (CFD) has predominantly been the methodology used to study IA hemodynamics. Yet, CFD assumptions and limitations coupled with the lack of CFD validation has precluded clinical acceptance of IA hemodynamic assessments and likely contributed to the contradictory results among previous studies. Experimental particle image velocimetry (PIV) studies have been noticeably limited in both scope and number among IA studies, in part due to the complexity associated with such experiments. Moreover, the limited understanding of the robustness of hemodynamic metrics across varying flow and measurement environments and the effect of transitional flow in IAs also remain open issues. In this work, techniques to enhance IA PIV capabilities were developed and the first volumetric pulsatile IA PIV study was performed. A novel blood analog solution—a mixture of water, glycerol and urea— was developed and an autonomous methodology for reducing experimental noise in velocity fields was introduced and demonstrated. Both of these experimental techniques can also be used in PIV studies extending beyond IA applications. Further, the onset and development of transitional flow in physiological, pulsatile waveforms was explored. The robustness of hemodynamic metrics such as wall shear stress, oscillatory shear index, and relative residence time across varying modalities, spatiotemporal resolutions, and flow assumptions was explored. Additional hemodynamic metrics which have been demonstrated to be influential in other cardiovascular flows but yet to be tested in IA studies were also identified and considered. Ultimately this work provides a framework for future IA PIV studies as well as insight on using hemodynamic evaluations to assess the risk of growth and rupture of an IA, thereby taking steps towards enhancing the clinical utility of such analysis.</p>

Es wurde die Bedeutung der intravaskulären Ultraschalluntersuchung (IVUS) bei Diagnostik (1) und interventioneller Therapie (2) der koronaren Herzerkrankung am Beispiel der Transplantatvaskulopathie (TVP) untersucht. 1. Bei 321 Patienten post-HTx-Patienten ohne relevante Stenosen im Koronarangiogramm wurde eine dreidimensionale Rekonstruktion des Ramus interventricularis anterior (LAD) und des linkskoronaren Hauptstammes aus den mittles manueller Katheterrückzugstechnik aquirierten IVUS-Bildern durchgeführt und die intrakoronare Plaqueverteilung und die mittlere Plaquelast der einzelnen Koronarsegmente analysiert. Bei 296 Patienten (92%) fanden angiographisch nicht sichtbare frühe Plaquebildung. 48% dieser Patienten zeigten ein fokal,polyfokale, 52% ein diffuses Plaqueverteilungsmuster. Unabhängige Prädiktoren für das Auftreten einer diffusen TVP waren männliches Geschlecht des Empfängers, das Zeitintervall zwischen HTx und IVUS-Untersuchung (Transplantationszeit) und das Spenderalter. In beiden morphologischen Untergruppen war ein häufigere und stärkere Plaquebildung in den proximalen Koronarsegmenten nachweisbar. Ein distaler Gefäßbefall war bei diffuser Plaquebildung signifikant häufiger und zeigte eine steigende Inzidenz mit zunehmender Transplantationszeit. Somit stellt das longitudinale Plaqueverteilungsmuster und der distale Gefäßbefall einen zusätzlichen morphologischen Marker für den Schweregrad einer beginnenden Transplantatvaskulopathie dar. 2. Bei 36 post-HTx Patienten wurden 62 Stenosen prospektiv mit einer IVUS-gesteuerten, gefäßgrößen-adaptierten Stentimplantation erfolgreich versorgt. Die Stentgröße wurde dem Mittelwert aus Lumen- und Gefäßdurchmessers im proximalen Referenzsegment angepaßt. Nach Vordilatation fand sich ein Lumengewinn von 1.26± 0.16 auf 1.95 ± 0.27mm, nach abschließender Stentimplantation auf 2.94 ± 0.37mm. Nach 6 Monaten betrug die binäre In-Stent-Restenosierungsrate 21.8%, eine Re-PTCA wurde bei 10.9 % durchgeführt. / The role of intravascular ultrasound imaging in diagnosis of coronary disease (1) and guiding percutaneous coronary intervention (2) was evaluated in patients with transplant vasculopathy. 1. In 321 post-HTx-patients without angiographic evidence of coronary disease, three-dimensional intravascular ultrasound imaging of the left anterior descending coronary artery (LAD) and the left main coronary artery was performed. Intracoronary plaque distribution and plaque burden was evaluated for each coronary segment. In 296 patients (92%) angiographically silent plaque was detected by IVUS. 48% of these patients showed a focal,polyfocal and 52% a diffuse plaque distribution pattern. Independent predictors of diffuse plaque formation were male gender of the recipient, transplantation time and donor age. In both morphological subgroups of plaque distribution the incidence and magnitude of plaque formation was highest in the proximal LAD segment. Plaque formation in the distal LAD was more frequent in diffuse plaque formation and increased significantly with time after transplantation. Therefore longitudinal plaque distribution pattern and distal vessel involvment are useful additional morphological markers for staging of beginning transplant vasculopathy. 2. In 36 post-HTx-patients 62 coronary stenosis were successfully treated by vessel-size adapted stenting by use of intravascular ultrasound guidance. The stent size was adapted to the proximal reference segment´s mean of lumen/vessel diameter. After pre-dilatataion the minimal lumen diameter increased from 1.26± 0.16 to 1.95 ± 0.27mm and to 2.94 ± 0.37mm after final stent implantation. After 6 months, binary in-stent-restenosis rate was 21.8% and target vessel revascularization rate 10.9%, respectively.

1981 entwickelten Gerd Binnig und Heinrich Rohrer bei IBM in Zürich das "Scanning Tunneling Microscope". Damit wurde erstmalig das lokal hochaufgelöste Erfassen (bis in den atomaren Auflösungsbereich) von Objekteigenschaften im Nahfeld inerter Oberflächen möglich. Dies und insbesondere die Weiterentwicklung der Technologie und die spätere (1986) Etablierung der Rasterkraftmikroskopie (Atomic Force Microscopy - AFM), die diese Auflösungsmöglichkeiten der Rastersondenmikroskope auch an Non-Konduktoren (nicht leitende Untersuchungsoberflächen) realisieren konnte, stellte die Geburtsstunde einer neuen mikroskopischen Ära auf dem Gebiet der biomedizinischen Grundlagenforschung dar (Kapitel 1.3). Das Studium der umfangreichen Literaturquellen zu diesem Thema und der direkte wissenschaftliche Kontakt und Erfahrungsaustausch mit anderen AFM- Arbeitsgruppen ließen im Initialstadium dieser vorliegenden Arbeit bereits erkennen, dass in der kardiovaskulären Grundlagenforschung zunehmend rasterkraftmikroskopische Versuchsansätze bearbeitet und kardiologisch interessante Fragestellungen mittels dieser Methode begleitend untersucht wurden (Kapitel 1.4). Das Ziel dieser vorliegenden Arbeit bestand darin, kardiovaskulär relevante Zellen und Einzelproteine in vivo und interventionelle Materialien (Stents) rasterkraftmikroskopisch zu untersuchen, wobei die Etablierung und technisch aufwendige Optimierung dieser neuen mikroskopischen (Kapitel 3.1) und der zellspezifisch präparatorischen Methoden (Kapitel 3.2) an diesen Untersuchungsobjekten im Mittelpunkt stehen sollte. Die im Rahmen dieser Arbeit untersuchten endothelialen Zellen und H9C2-Myozyten stammten aus, in unserem Forschungslabor etablierten, immortalen Kulturzelllinien. Die adulten und Kardiomyozyten neonataler Ratten, die kardial- fibrozytären Zellen sowie die Thrombozyten wurden primär isoliert und als Primärkulturzellen kultiviert (Kapitel 3.2.3 und 3.2.4). Außerdem wurden vitale aortale Endothelzellen unterschiedlicher Tiere (Ratte, Meerschwein, Kaninchen) im Gewebsverband der thorakalen Aorta untersucht (Kapitel 4.2). Die Zellen wurden initial, im Rahmen der Etablierungsphase mittels unterschiedlicher Methoden fixiert und nachfolgend rasterkraftmikroskopisch untersucht und dargestellt. Der Etablierungsprozess der Methodik begann mit der Abbildung luftgetrockneter Zellen (Kapitel 4.1.1) unter Raumbedingungen und setzte sich über verschiedene Modifikationen der Zellpräparation (z.B. Glutardialdehydfixation, Cryofixation), des Abbildungsmodus (Contact-, Non-Contact-, Tapping-Mode) und der Abbildungsbedingungen (Raumbedingungen, zellphysiologische Umgebung) fort, so dass schließlich die Abbildung vitaler Zellen (Kapitel 4.1.2 und Kapitel 4.2 - 4.5) in ihrer strukturellen und funktionellen Umgebung (z.B. aortale Endothelzellen im Gewebsverband) etabliert werden konnte und routinemäßig reproduzierbar war. An stabilen oder künstlich stabilisierten Strukturen der o.g. vitalen Zellen wurden erste orientierende Messungen der bioelastischen Eigenschaften (Kraft-Abstands-Kurven, Kapitel 4.1.2.1) durchgeführt. Außerdem haben wir im Einzelfall, wenn technisch und apparativ möglich, andere hochauflösende strukturanalytische Verfahren (z.B. TEM) als mikroskopische Referenzuntersuchungen herangezogen (Kapitel 4.1.2; 4.4.1; 4.6), wobei z.T. erstaunliche Übereinstimmung zwischen den AFM- Daten und den strukturanalytischen Daten der Referenzmethoden nachweisbar waren. Ein strukturell durch Elektronenmikroskopie und Röntgendiffraktionsanalyse sehr gut beschriebenes komplexes Funktionsprotein, das 20-S-Proteasom, wurde mittels der Rasterkraftmikroskopie abgebildet und vermessen und die so gewonnenen strukturanalytischen Daten mit den bekannten strukturellen Abmessungen des Proteins verglichen (Kapitel 4.6). Die hierbei detektierten dimensionalen Abweichungen zwischen den AFM- assoziierten Daten und den bekannten strukturanalytischen Daten der Elektronenmikroskopie wurden im Kontext der funktionellen Integrität des Proteins und hinsichtlich möglicher methodischer Fehlereinflüsse (Kapitel 3.1.4.3) diskutiert. Interventionelle Materialien (Stents), die in der täglichen kardiologischen Praxis Anwendung finden, sind hinsichtlich ihrer Ultrastruktur mittels dieser hochsensitiven Abbildungsmethode im Nahfeld von Objektoberflächen untersucht worden. Bezüglich ihrer nativen Oberflächenbeschaffenheit und ihrer mechanischen Alteration durch den Ballon- Dilatationsprozess wurden die Stents sehr detailliert qualitativ und quantitativ (Kapitel 4.7) beschrieben, wobei Prädilektionsstellen der prozedural- assoziierten mechanischen Beanspruchung der Stents durch die hier beschriebene, oberflächensensitive AFM- Methode sehr genau diskriminiert werden konnten. Die präparierten Stents wurden weiterführend mit humanen Thrombozytenkonzentraten inkubiert und die Zell- Stentoberflächenkontakte sowie mögliche Stentoberflächen- induzierte Veränderungen der Thrombozyten sind morphologisch ausführlich beschrieben worden. Letztendlich wurde im Rahmen der vorliegenden Arbeit die spezifische Aktivierung der vitalen Thrombozyten durch pharmakologische Stimulantien (z.B. ADP) mit der, durch den AFM-Abbildungsprozess induzierten Thrombozytenaktivierung (Kapitel 4.5) unter AFM-Bedingungen verglichen und diskutiert. Die Ergebnisse dieser Arbeit weisen, dass mit der AFM-Technologie und objektorientiert optimierten Mess- und Präparationsmethoden ein neues mikroskopisches Analyseverfahren vorliegt, dass zum einen real-dreidimensionale morphologische Bildgebung bis in den submolekularen Auflösungsbereich an vitalen Zellen und präparierten Proteinkomplexen, zum anderen aber gleichermaßen Funktionsanalytik in Form von Messungen zelldynamischer Prozesse wie Migrationsbewegungen und Kontraktionen sowie visko- elastische Quantifizierung von Zellmembranen erlaubt. Der Vorteil gegenüber den meisten gegenwärtig verfügbaren mikroskopischen Methoden liegt in der neu eröffneten Möglichkeit der seriellen, wiederholten und stabil reproduzierbaren Messung an vitalen Zellen und zellulären Substrukturen. Insofern könnte in Zukunft diese neue Technologie eine methodische Bereicherung der mikroskopisch-morphologisch und funktionell orientierten Analysetechnik darstellen. / In 1981 Binnig and Rohrer invented the "Scanning Tunneling Microscope". Thereby it became feasible to high-resolution record the surface-properties of specimens (up to atomic resolution) at the nearfield of inert surfaces. This and in detail the further development of this technology and the establishment of "Atomic Force Microscopy" (1986), that allows implementation of this resolution capabilities in non-conductors or insulating materials represent the birth of a new microscopic era in the field of biomedical basic research (chapter 1.3). The promise of atomic (scanning) force microscopy (AFM) for cardiovascular research is enormous. The perusal of the extensive literature concerning this topic and scientific contact with other researchers reveals initial the capabilities of this method in cardiovascular basic research. Intriguing questions of cardiology may investigate concomitantly with help of scanning-force-micoscopic approaches (chapter 1.4). The aim of this study was to investigate relevant cardiovascular cells and single proteins in-vivo and specific materials (coronary artery stents) with scanning-force-micoscopic setup. The establishment and expensive optimization of this new microscopic method (chapter 3.1) and of the cell specific preparatory methods (chapter 3.2) represented the center of interest of our inevestigations. The endothelial cells and H9C2-myocytes stem from established imortal cell culture lines. The adult cardiomyocytes and cardiomyocytes of neonatal rats, the fibrocytes and the thrombocytes were primarily cultivated (chapter 3.2.3 and 3.2.4). In addition we investigated aortic endothelial cells of intact aortic tissue of different animals (rat, guinea pig, rabbit - chapter 4.2). During the establish experiments cells underlied different methods of cell-fixation. The primary investigations was performed using air-dried cells (chapter 4.1.1) analyzed in room ambient conditions and were continued by different modifications of cell-preparation. (e.g. glutardialdehyde-fixation, cryo-fixation), of microscopic mode (contact-, non-contact-, tapping-mode) and of cell-specific environmental conditions (from room ambient to cellphysiological medium and temperature). As result we became enabled to investigate (reproducible and routinely) vital cells (chapter 4.1.2 and chapter 4.2 - 4.5) embedded in physiological normal structural und functional ambient conditions (e.g. endothelial cells of intact aortic tisue in-vivo). Additionally, we performed measurements of bio-elastic properties of stable or artificial stabilized structures of named cells (force-distances-curves - chapter 4.1.2.1). If posibble, depending of available technical equipment, we compared our microscopic results with other high-resolution analytical procedures of reference (e.g. TEM - Kapitel 4.1.2; 4.4.1; 4.6) and detected astonishing congruence between the data. Furthermore we analyzed the well-described (electron-microscopy and x-ray-diffraction data) complex 20-S-proteasome using a specific atomic force microscopic setup. Analytical and structural data of these AFM-scans and abovementioned methods were likened (chapter 4.6). The deviations concerning the detected proportions were discussed regarding functional integrity of the protein and with respect to potential methodically determined artifacts. (chapter 3.1.4.3). Assaying (qualitative and quantitative) the surface roughness properties of coronary artery stents, we found significant alterations of stent material induced by balloondilatation. We suppose, that changes in roughness of inner surface of coronary artery stents might induce clinical problems like acute stent-thrombosis and in-stent-restenosis. Finally these stents were coated with human thromboytes to investigate cell-stent-surface interactions. Surface-roughness correllated triggering of thrombocyte adhesion was evaluated by morphological analysis of AFM-scans. Finishing, we have investigated and concluding discussed the specific activation of vital thrombocytes by pharmacological substances (e.g. ADP) and by mechanical stimulation (due to AFM-associated tip-surface-interaction). The results of this work demonstrate, AFM-technology using optimized microscopic setup and object-specific adjusted measurement- and preparation- methods, is an new, powerful, microscopic technique, that allow real-3-dimensional morphological mapping up to submolecular range of resolution in vital cells and protein complexes. Moreover, this technology opens new dimensions in functional analytic of cell migration processes or cellular contractions and in evaluation of visco-elastic quantification of cell membranes. The advantage owed to the most currently available microscopic methods is the option of serial and reproducible measurement of vital cells and subcellular structures. In this respect, this new method might represent a methodical enrichment of the microscopic-morphological and functional oriented analysis-technique in future.